The suppressive function of pltLys-iTregs in the immune response in vivo. One million CD4⁺EGFP⁺ Treg cells (iTregs/CD45.1⁺) induced in vitro by pltLys or purTGFβ were sorted and transfused into F8^null/CD45.2⁺ mice followed by rhF8 immunizations. (A) Schematic diagram of iTreg cells and rhF8 transfusion in F8^null(B6) mice. (B) Stability of Foxp3 expression on Treg cells induced by pltLys. Foxp3^EGFP expression was analyzed by flow cytometry on peripheral CD45.1⁺CD4⁺ T cells 6 days after iTreg cell transfusion. (C) The incidence of anti-FVIII inhibitor development. Bars represent percentages. (Di) The titers of inhibitory antibodies in immunized F8^null mice as determined by Bethesda assay. The Kruskal-Wallis test was used to compare the 3 groups, and the Mann-Whitney U test was used for pairwise comparison. (Dii) The inhibitor titers in immunized F8^null mice that are ≤100 BU/mL. This is a zoom-in look at the lower part of panel Di. (E) The incidence of total anti-FVIII antibody development. Bars represent percentages. (F) The total anti-FVIII antibody titers in immunized F8^null mice as determined by ELISA. The Kruskal-Wallis test was used to compare the 3 groups, and the Mann-Whitney U test was used for pairwise comparison.